Synucleins in Glaucoma: Implication of�-Synuclein in Glaucomatous Alterations inthe Optic Nerve
Irina Surgucheva,1 Belinda McMahan,1 Farid Ahmed,2 Stanislav Tomarev,2
Martin B. Wax,1 and Andrei Surguchov1*1Department of Ophthalmology and Visual Sciences, Washington University, St. Louis, Missouri2Laboratory of Molecular and Developmental Biology, National Eye Institute, National Institutes of Health,Bethesda, Maryland
The synucleins are a family of soluble proteins thatare widely expressed in neurons and other cell types of thecentral nervous system. Recent studies suggest thatsynucleins contribute to the pathophysiology of severehuman illnesses, including neuronal degenerations.�-Synuclein is known to be a component of the filamentsin Lewy bodies in Parkinson’s disease (PD), dementia with
Lewy bodies (Spillantini et al., 1997, 1998; Baba et al.,1998), and glial cytoplasmic inclusions in multiple systematrophy (Tu et al., 1998; Dickson et al., 1999). AnAla53Thr mutation in the �-synuclein gene has beenproved to cause a familial type of PD (Polymeropoulos etal., 1997). In addition, a second type of missense mutation,Ala30Pro, has been reported in familial PD (Kruger et al.,1998).
The synucleinopathies are a diverse group of neuro-degenerative diseases with a similar pathologic lesion com-posed of aggregates of insoluble �-synuclein protein inselectively vulnerable populations of neurons and glia.Growing evidence links the formation of abnormal fila-mentous aggregates to the onset and progression of clinicalsymptoms and the characteristic lesions in affected brainregions of patients with neurodegenerative disorders.
�-Synuclein is a small acidic protein composed of140 amino acid residues. It has seven incomplete repeats of11 amino acids with a core of KTKEGV, whereas theC-terminal portion has no known structural elements.�-Synuclein originally isolated from the bovine brain ishighly homologous to �-synuclein. Because of the similarlocalization of �- and �-synucleins proteins, predomi-nantly in the presynaptic terminals of neurons, it has beenspeculated that they are involved in synaptic function.�-Synuclein was isolated from breast cancer tissue andinitially termed “breast cancer-specific gene 1” (BCSG1; Jiet al., 1997). �-Synuclein is expressed in the brain (Galvin
Contract grant sponsor: NJH NEI; Contract grant number: EY 13784;Contract grant sponsor: The Glaucoma Foundation; Contract grant num-ber: QB42308; Contract grant sponsor: Carl Marshall Reeves and MildredAlmen Reeves Foundation; Contract grant sponsor: ADRC; Contractgrant number: 99-6403.
*Correspondence to: Andrei Surguchov at his current address, 4004 Ha-worth Hall, Kansas University, Lawrence, KS 66045.E-mail: [email protected]
Received 29 October 2001; Revised 26 December 2001; Accepted 28December 2001
Published online 6 March 2002 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jnr.10198
et al., 1999) and spinal cord, in addition to breast tissue(Lia et al., 1999), but is most abundant in the peripheralnervous system (Buchman et al., 1998a; Ninkina et al.,1998, 1999; Lavedan et al., 1998).
In spite of the growing number of publications onsynucleins and their established involvement in neurode-generative diseases, their presence in ocular cells and tis-sues and possible role in eye diseases was not studied untilrecently (Surguchov et al., 1999, 2001a). We have shownthat all three members of the synuclein family (�, �, and�) are differentially expressed in the retina and optic nerveand that the expression pattern for some members of thefamily changes significantly in the retinas of Alzheimer’sdisease (AD) patients and in transgenic mice overexpress-ing �-synuclein (Surguchov et al., 2001a).
Glaucoma, the second leading cause of permanentvision loss in the world (Quigley, 1996), is a group ofocular disorders that are responsible for the excavation andatrophy of the optic nerve, loss of retinal ganglion cells,and gradual loss of visual field (Quigley, 1993; Friedmanand Walter, 1999). The pathogenesis of this disease is farfrom being completely understood. Glaucoma is usuallyconsidered as a neurodegenerative disease, and there aresome studies proposing possible common mechanismsand/or common components with neurodegenerative dis-eases of the brain, e.g., AD (Vickers et al., 1995a, b; 1997).Other investigators, although accepting common symp-toms and mediators of toxicity for these two diseases,consider that they have distinct temporal, subcellular, andsignal-transduction features (Schwartz et al., 1999). Inaddition, glaucoma is usually, but not always, associatedwith elevated intraocular pressure (IOP).
Here we demonstrate considerable changes in thelocalization of �-synuclein in the optic nerve of glaucoma
patients and in an animal model of glaucoma. In addition,incubation of astrocytes at elevated hydrostatic pressurecauses a reduction in the amount of �-synuclein. Thesedata suggest that �-synuclein may be implicated in theglaucomatous changes that occur in the optic nerve.
MATERIALS AND METHODS
Tissue Preparation
Sagittal paraffin-embedded 6 �m human tissue sectionswere a gift of Dr. M.R. Hernandez (Washington University, St.Louis, MO). The sections were prepared as described earlier(Agapova et al., 2001). Postmortem human eyes from 16 donorswere analyzed; among the donors, 10 had been diagnosed withdifferent forms of glaucoma. Age of the ocular tissue donorsranged from 61 to 87 years (Table I). Six human donor eyeswithout history of eye disease or neurodegenerative disease wereused as age-matched controls.
The eyes were obtained from the Glaucoma ResearchFoundation (San Francisco, CA), from the Mid-America EyeBank (St. Louis, MO), and from Dr. Martin B. Wax (Washing-ton University, St. Louis, MO). Clinical findings of the glau-coma patients were well documented during 5–13 years offollow-up period, including IOP readings and optic disc andvisual field changes. There was no diabetes, collagen vasculardisease, infection, or sepsis in any of the donors. The cause ofdeath for all of the donors was acute myocardial infarction orcardiopulmonary failure. The eyes were enucleated within 2–4hr after death and processed and fixed within 6–12 hr in either10% buffered formaldehyde or 4% paraformaldehyde. The pos-terior poles were dissected free of surrounding tissues, washedextensively in 0.2% glycine in phosphate-buffered saline (PBS)at pH 7.4, embedded in paraffin, and oriented sagittally for 5 �msections.
TABLE I. Clinical Data on Postmortem Glaucomatous Eyes
Patient number Age (years) Diagnosis IOP
Control group Normal1 84 No glaucoma symptoms2 66 No glaucoma symptoms3 68 No glaucoma symptoms4 74 No glaucoma symptoms5 85 No glaucoma symptoms6 74 No glaucoma symptoms
Antibody to �-synuclein (G-syn AB01) was raised andaffinity purified by reacting with immunizing peptide immobi-lized on cyanogen bromide-activated Sepharose 4B as describedpreviously (Surguchov et al., 2001a). This antibody was diluted1:3,000–1:4,000 for immunohistochemical (IHC) staining. Incontrol experiments with peptide antigen, no staining was ob-served. For experiments with rat samples, we also used com-mercial antibody to �-synuclein raised against a peptide corre-sponding to amino acid 114–127 of the human �-synuclein(ABCam U.K.). This antibody cross reacts with rat �-synuclein.It was diluted 1:750 for Western blot and 1:3,000 for IHCstaining. We used, as antibody for �-synuclein, rabbit or sheeppolyclonal antiserum produced by Chemicon International Inc.(Temecula, CA), dilution 1:1,000 for IHC staining. Affinity-purified polyclonal rabbit anti-�-synuclein antibody was pur-chased from Chemicon International Inc. and was diluted1:5,000 for IHC staining. Biotinylated secondary antibody wasidentified by reacting with the streptavidin-peroxidase conju-gate (Vector Laboratories, Burlingame, CA). For Western blot,secondary antibodies were conjugated with peroxidase, dilution1:50,000 (Amersham, Buckinghamshire, United Kingdom). For�-tubulin, we used monoclonal anti-�-tubulin DM 1A anti-body (Sigma, St. Louis, MO) diluted 1:5,000 and, for actin,monoclonal anti-actinN350 antibody (Amersham) in dilution1:2,500.
Immunohistochemical Staining of the Optic Nerve
Human or rat eye tissues were paraffin embedded, andslices of 6 �m were cut and placed on silane-coated slides.Before immunostaining, slides were deparaffinized and incu-bated for 1 hr in PBS glycine at room temperature to reducenonspecific binding. Heat-induced epitope retrieval procedurewas used for both human and rat slices. We followed theprotocol of epitope retrieval described by Zymed (South SanFrancisco, CA). Slides were preincubated with 5% milk for30 min, rinsed, and incubated with primary antibodies for30 min. The slides were washed in PBS/Triton X-100 threetimes for 5 min each, with a subsequent wash in PBS for 5 min.Biotinylated secondary antibody was placed on the sections andincubated for 30 min, washed with PBS, and reacted with thestreptavidin-peroxidase conjugate (Vector Laboratories) for30 min. The bound antibody-peroxidase complexes on thesections were visualized using a 3,3-diaminobenzidine tetrahy-drochloride (DAB) substrate solution consisting of 1.5 mg DABand 50 �l 30% hydrogen peroxide, in 10 ml 0.1 M Tris, pH 7.6.The sections were incubated in the dark until brown stainingappeared, washed in PBS, counterstained with hematoxylin,dehydrated, and coverslipped with Permount. Control sectionswere run in parallel, omitting only the primary antibody.
A7 Astrocytes From Rat Optic Nerve
We used immortalized rat central nervous system cells ofprimary cultures of rat optic nerve A7 (gift of Dr. H. Geller).The culture was immortalized with murine leukemia virus psi-2,SV-40-6, which is defective in assembly and contains the SV-40large T antigen and neomycin-resistance genes, as describedearlier (Geller and Dubois-Dalcq, 1988). This stable immortal-ized clonal cell line expressed nuclear SV-40 large T cells and the
astrocyte-specific marker glial fibrillary acidic protein (GFAP).The cells were grown as described previously (Geller andDubois-Dalcq, 1988).
Use of Hydrostatic Pressure
A pressure chamber equipped with a manometer was usedaccording to the protocol described previously (Ricard et al.,2000). Briefly, the A7 cells were split on the glass coverslips in35 mm Petri dishes in Dulbecco’s modified Eagle’s medium(DMEM) containing 10% fetal bovine serum (FBS). The cellswere grown at 37°C in 5% CO2 until they reached 40% con-fluence. Then, the experimental plates were put in the chamber,where the hydrostatic pressure was raised to 60 mm Hg aboveambient pressure. The chambers were placed in a tissue cultureincubator and maintained at 37°C. Water was placed inside thechamber to maintain 97% relative humidity. The control plateswere cultured under similar conditions at normal atmosphericpressure.
Immunocytochemical Staining
For single and double staining of primary culture of ratastrocytes, the cells were split on glass coverslips at �50–60%confluence and kept at 37°C in 5% CO2. Cells were washed inPBS and fixed with 100% methanol for 10 min at –20°C, thenwith a methanol:acetone mixture (1:1) for 4 min at –20°C. Afterintensive washing in PBS, the samples were blocked for 1 hr in1% bovine serum albumin (BSA) in PBS with 0.1% TritonX-100 at room temperature. For single or double staining, thecoverslips were placed facing downward on a drop of primaryantibody (20–40 �l) diluted in blocking solution and left in ahumidified chamber at 4°C overnight. After washing with PBSthree times for 7 min each, coverslips were incubated with goatanti-rabbit Oregon green-conjugated or goat anti-mouse rho-damine red-conjugated secondary antibody. After the finalwash, coverslips were mounted in Vectashield (Vector Labora-tories). Fluorescent images were taken using an Olympus BH-2fluorescence microscope (Olympus Japan) equipped with a 568nm filter for rhodamine red and a 488 nm filter for Oregongreen. Images were recorded by digital photography (Spot Di-agnostic Instruments) and stored as computer files.
Rat Model of Glaucoma
All experiments complied with the guidelines published inthe NIH Guide for the Care and Use of Laboratory Animals(National Institutes of Health Publication No. 8523) and theARVO Statement for the Use of Animals in Ophthalmic andVision Research. Twelve animals were used in these experi-ments. The experiments were performed with interval of twomonths. With the animal under anesthesia (mixture of 45 mg/kgof ketamine and 9 mg/kg of xylazine), IOP was elevated in theleft eye of the adult female albino Wistar rats by cauterizing threeepiscleral veins as previously described (Shareef et al., 1995).The right eye served as a sham-operated control. The IOP valuewas measured with a precalibrated Mentor pneumatonometer(Bio-Rad, Hercules, CA). Those rats in which IOP returned tonormal after 2–4 weeks were excluded from the study. Theremaining rats were sacrificed approximately 6 weeks after theoperation and used for preparation of optic nerve samples forIHC staining and Western blot analysis.
Synucleins in Glaucoma 99
Semiquantitative RT-PCR
One microgram of total RNA was used for cDNA syn-thesis using Superscript reverse transcriptase (Gibco BRL, GrandIsland, NY) and oligo(dT)-primer. cDNA samples were dilutedto provide a linear range of PCR. After adjustment of cDNAconcentration for each individual pair of samples from thecontrol and experimental eyes of the same animal, relativeabundances of mRNA for �-synuclein were estimated. Thefollowing primers were used: for �-Synuclein, forward primer5�- ggaggccaaagagcaagagga-3� and reverse primer 5�-agcgtctggaaggtgatccgaa-3�, product size 282 bp; primers for�-synuclein were located in different exons; for cyclophilin,forward primer 5�-tcctcctttcacagaattattcc-3� and reverse primer5�aattagagttgtccacagtcgg-3�, product size 345 bp. PCRs wereperformed in a PTC-200 Thermal Cycler (MJ Research, Wa-tertown, MA) using AmpliTaq polymerase (Perkin-Elmer, OakBrook, IL) as described previously (Ahmed et al., 2001). EachPCR was repeated at least twice. After initial denaturation for 90sec at 94°C, the following conditions were used for amplifica-tion: 30 cycles of 30 sec at 94°C, 90 sec at 59°C, 60 sec at 72°C,and a final incubation for 5 min at 72°C. PCR products wereanalyzed by agarose gel electrophoresis. Gels were stained byethidiun bromide, and the intensity of DNA bands was esti-mated using Chemilimager 4000 software (Alpha Innotech Inc.,San Leandro, CA). Our previous experiments with the Myoc/Tigr gene demonstrated that, under the conditions used in ourexperiments, semiquantitative RT-PCR provided reliable esti-mates of the changes in the level of analyzed mRNA that areconfirmed by real-time PCR and northern blot hybridization(Ahmed et al., 2001).
Western Blots
Total protein was measured by the Bradford method(Pierce, Rockford, IL). Rat brain was a gift of Dr. C. Romano(Department of Ophthalmology, Washington University, St.Louis, MO). Total protein extract (15–20 �g) was loaded on a12% polyacrylamide gel. After electrophoresis, proteins weretransferred onto nitrocellulose membane at 0.45 �M (Bio-Rad).Nonspecific binding sites were blocked by immersing the mem-brane in 5% blocking reagent in Tris-buffered saline Tween(TBS-T) for 1 hr at room temperature on an orbital shaker.Membranes were washed, incubated with antibody, and ex-posed to the film as described by the manufacturers of ECLWestern blotting detection reagents (Amersham Pharmacia Bio-tech). To quantitate the squares of the peaks, the films werescaned by gel scanner AlphaImager 2200 (Alpha Innotech Inc.)using the software AlphaEase v5.5.
RESULTS
Synuclein Localization in Human Retina andOptic Nerve of Glaucoma Patients
We analyzed the distribution of three members ofthe synuclein family in ocular tissues of donors with dif-ferent forms of glaucoma. No significant differences in thelocalization of �- and �-synuclein between normal andglaucomatous patients were found (not shown). For allsamples, we observed that the pattern of immunopositivestaining for these two members of the synuclein family was
identical or similar to the pattern described in our previouspublication (Surguchov et al., 2001a). However, the lo-calization of �-synuclein in glaucomatous optic nervechanges significantly both in the area of lamina cribrosaand in the postlamina area. As shown in Figure 1A,B,�-synuclein is present in nerve bundles and practicallyabsent in glial cells in the area of lamina cribrosa in controleyes. In the optic nerves of donors with primary open-angle glaucoma (POAG), chronic-angle-closure glaucoma(COAG), or normal-tension glaucoma (NTG) and someglaucoma patients without history of the disease, in addi-tion to nerve bundles, a strong immunopositive staining ofglial cells was seen (Fig. 1C–F, arrows). Similar changeswere found in post lamina area of the optic nerve (Fig. 2).Strong �-synuclein-immunopositive staining of inclusionsin glial cells accompanied by a reduction in the staining ofnerve bundles was also observed for a patient with pseudo-exfoliative form of glaucoma (Table I, patient 16; notshown). In more advanced cases of disease, when nervebundles are heavily disorganized, the appearance of strong�-synuclein-immunopositive inclusions becomes moreevident.
We also observed some differences in the stainingpattern of synucleins in retinas of glaucoma patients com-pared with age-matched controls. For example, weakstaining by �-synuclein of the inner plexiform layer wasobserved, whereas both layers were immunonegative for�-synuclein in control samples (not shown). To gain adeeper insight into the effect of elevated pressure on thelocalization of �-synuclein in the optic nerve, we used ananimal model of glaucoma.
IHC Staining of the Optic Nerve of Rats Used as aModel of Glaucoma
In the optic nerves of both control and experimentalanimals, nerve bundles are immunopositive for �- and�-synucleins throughout the optic nerve (not shown).The staining of some processes of glial cells is also ob-served. This pattern of staining is similar to the patterndescribed previously for human optic nerve (Surguchov etal., 2001a). We did not observe significant changes in �-and �-synuclein immunoreactivity under experimentalanimals compared with controls.
The majority of �-synuclein in the optic nerve ofcontrol eyes is also localized in the nerve bundles (Fig. 3A,arrows). At the same time, in the optic nerve of experi-mental rats with high IOP, �-synuclein-immunopositiveglial inclusions were observed, whereas the intensity ofnerve bundle staining was reduced (Fig. 3B).
Effect of Experimental IOP Elevation on �-Synuclein mRNA Level in Rat Optic Nerve Head
To gain insight into mechanisms involved in theregulation of �-synuclein after experimental induction ofelevated IOP, levels of �-synuclein mRNA were evalu-ated in the optic nerve of experimental and control ani-mals by semiquantitative PCR (Fig. 4). In the optic nerveof experimental animals with elevated IOP, the level of�-synuclein mRNA is slightly reduced [for example, com-
100 Surgucheva et al.
pare Fig. 4, lane 3 (control), with lane 4 (experimental eye)and lane 5 (control) with lane 6 (experimental eye)]. Themost significant reduction was observed 4 weeks afteroperation (reduction to 73% compared with controls).The difference between control and experimental sampleswas statistically significant (P � 0.032). No significantchanges in the level of cyclophilin mRNA used as acontrol were observed in experimental animals (Fig. 4,bottom).
Effect of Elevated Pressure on the Amount of �-Synuclein in Neural Ocular Tissues
According to Western blot analyss, the amount of�-synuclein was reduced in the optic nerve of experimen-tal rats (59% � 4% compared with control samples, aver-age of three experiments; Fig. 5A, lanes 4, 5), whereas no
significant changes were found in the retina of experimen-tal animals compared with control samples (Fig. 5A, lanes2, 3). At the same time, elevated IOP and elevated hy-drostatic pressure did not affect the amount of actin in theoptic nerve of experimental animals (Fig. 5C, lanes 4, 5) orin astrocytes (Fig. 5C, lanes 7, 8) but reduced the amountof �-tubulin both in the optic nerve of experimentalanimals (Fig. 5B, lanes 4, 5) and in astrocytes incubated atelevated hydrostatic pressure (Fig. 5B, lanes 6, 7). Unlikethe case with the optic nerve and astrocytes, no effect ofelevated IOP on the amount of �-synuclein, �-tubulin,and actin in the retina was found (Fig. 5A, lanes 2, 3, for�-synuclein; Fig. 5B, lanes 2, 3, for �-tubulin; Fig. 5C,lanes 4, 5, for actin).
The reduction of �-synuclein was also observed afterimmunohistochemical staining of astrocytes incubated un-
Fig. 1. Sections of the human opticnerve in the area lamina cribrosa.�-Synuclein was detected using DAB re-agent (peroxidase, brown); samples werecounterstained with hematoxylin (blue).Sections were stained with G-synAB-01antibody as described in Materials andMethods. A,B: Samples from controlindividuals (samples 1 and 2, respec-tively, in Table I). C: Sample from apatient with CACG (patient 11, TableI). D: Sample from a patient with NTG(patient 12, Table I). E: Sample from apatient with POAG (patient 7, Table I).F: Patient with glaucoma without his-tory of disease (patient 14, Table I). Ar-rowheads in A and B indicate nervebundles, and arrows in C–F show im-munopositive staining in glial cells. Scalebar � 10 �m.
Synucleins in Glaucoma 101
der elevated hydrostatic pressure (Fig. 6A,B). The majorityof �-synuclein-immunopositive staining in astrocytes waslocated in the nucleus, and a fraction of it was associatedwith intracellular structures located near the nucleus,which were previously identified as centrosomes (Surgu-chov et al., 2001b; Fig. 6, arrows).
DISCUSSIONThe synucleinopathies are a diverse group of neuro-
degenerative disorders that share a common pathologiclesion composed of aggregates of insoluble synucleins inselectively vulnerable populations of neurons and glia(Galvin et al., 2001). Most papers describing synuclein-related pathologies concern �-synuclein. The role ofsynucleins in neurodegenerative diseases is well establishedfor brain tissues but has not been characterized in detail for
different ocular pathologies in the retina and optic nerve.This is the first study to characterize the expression ofthree members of the synuclein family of proteins in tissuesand cells of ocular origin in connection with glaucoma. Asshown in Figures 1 and 2, we found abnormal IHCstaining for �-synuclein in the glaucomatous optic nervetissues. For patients with different types of glaucoma, weobserved �-synuclein-positive staining in a subset of glialcells, presumably reactive astrocytes, whereas, in the opticnerves of control individuals, the staining of nerve bundlesprevailed. However, we have not seen noticeable changesin the pattern of �- and �-synuclein staining in glauco-matous optic nerves compared with controls.
To clarify whether elevated pressure may be a factorregulating the level of �-synuclein, we used two modelsystems, and animal model in which elevated IOP in the
Fig. 2. Longitudinal sections of the hu-man postlamina part of the optic nerve.�-Synuclein was detected using DAB re-agent (peroxidase, brown); samples werecounterstained with hematoxylin (blue)as described in Materials and Methods.Sections were stained with G-synAB-01antibody. A,B: Samples from control in-dividuals (samples 1 and 2, respectively,in Table I). C: Sample from a patientwith CACG (patient 11, Table I).D: Sample from a patient with NTG(patient 12, Table I). E: Sample from apatient with POAG (patient 7, Table I).F: Patient with glaucoma without his-tory of disease (patient 14, Table I). Ar-rowheads in A and B indicate nervebundles, and arrows in C–F show im-munopositive staining in glial cells. Scalebar � 10 �m.
102 Surgucheva et al.
eye was generated by a cauterization of episcleral veins(Shareef et al., 1995) and a cell model in which the effectof hydrostatic pressure on astrocytes was analyzed in apressurized chamber (Ricard et al., 2000). According toWestern blot analysis, elevated hydrostatic pressure causeda reduction of �-synuclein level in the optic nerve andastrocytes but did not affect its amount in the retina. Thereduction of �-synuclein as a result of elevated pressureshows that it does not behave as the majority of stressproteins or heat shock proteins, the expression of which isup-regulated as a result of stress. At the same time, char-acteristic lesions were observed both in human glaucoma-
tous optic nerve (Figs. 1, 2) and in the optic nerve of ratswith high IOP (Fig. 3) as glial inclusions immunopositivefor �-synuclein.
The changes in �-synuclein immunoreactivity in theoptic nerve that we observed in the animal model inresponse to elevated IOP are slow compared with thereaction of glial cells to elevation of IOP described in ratretina by Wang and coworkers (2000). These authorsobserved rapid and synchronous reactivity of glial cellswithin hours after elevation of IOP, which is supposedlylinked to neuronal degeneration.
The appearance of glial inclusions immunopositivefor �-synuclein in the optic nerve of glaucoma patientsand in an animal model of glaucoma (Figs. 1–3) may beconsidered as a histopathological hallmark of glaucoma-tous alterations. Glial cells are key elements in the dynamicenvironment of neurons, forming a functional unit in-volved in homeostasis, plasticity, and neurotransmission(Ridet et al., 1997). Astrocytes, the major cell type in theoptic nerve head, are most intimately involved in thereaction to different stresses in the optic nerve, includingIOP and ischemia. One of the roles of reactive astrocytesis the preservation of neural tissue integrity from differentinjuries. Astrocytes of the optic nerve head have beenshown to be reactive in glaucomatous eyes (Rao andLund, 1993; Hernandez and Pena, 1997). Activated astro-cytes and microglia alter the microenviroment of neurons.Reactive astrocytes are responsible for generating a glialscar that limits the area of damage. At the same time,
Fig. 4. mRNA level for �-synuclein (top) and cyclophilin (bottom)measured by semiquantitative RT-PCR in the optic nerve of rats afterelevation of IOP. Lanes 1–4: Four weeks after cauterization. Lanes5–10: Five weeks after cauterization of three episcleral veins. Lanes 1,5: RNA isolated from a control eye C1; lanes 2, 6: RNA isolated fromexperimental eye E1; lanes 3, 7: RNA isolated from control eye C2;lanes 4, 8: RNA isolated from experimental eye E2; lanes 9, 10: RNAisolated from control eye C3 and experimental eye E3, respectively.RT-PCR was carried out as described in Materials and Methods and ina previous paper (Ahmed et al., 2001).
Fig. 3. Longitudinal section of the ratoptic nerve. A: Sample from a controleye; arrows show nerve bundles.B: Sample from an experimental eye;arrows show �-synuclein-immuno-positive glial inclusions. �-Synuclein wasdetected using DAB reagent (peroxidase,brown); samples were counterstainedwith hematoxylin (blue). Sections werestained with commercial antibody (seeMaterials and Methods).
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reactive astrocytes may damage axons of retinal ganglioncells through the release of neurotoxic agents and media-tors (Hernandez and Pena, 1997). In spite of extensivestudies of astrocytes and microglia during the past several
years, their role in normal eye tissues and in glaucomatousoptic nerve is still under debate.
The changes in the localization of �-synuclein im-munoreactivity that we have found in human glaucoma-tous optic nerve and in the optic nerve of experimentalanimal model are most probably the consequences ratherthan a cause of pathological changes that occur in glau-coma. However, we cannot exclude a more direct role for�-synuclein in the etiology of this disease. Several putativemechanisms may be involved. 1) This role may be con-nected with the �-synuclein effect on neurofilament net-work integrity described recently. Injection of�-synuclein-expressing vector in cultured sensory neuronscaused a dramatic decrease in triplet neurofilament protein
Fig. 5. Effect of elevated pressure on the amount of �-synuclein (A),�-tubulin (B), and actin (C) in rat tissue samples analyzed by Westernblotting. A: Extracts from rat brain (lane 1), retina (lanes 2, 3) or opticnerve (lanes 4, 5) were prepared as described previously (Surguchov etal., 2001a,b) and subjected to electrophoresis in 12% PAGE, Westernblotted, and probed with antibody to �-synuclein (ABCam U.K.). Lane2: Retinal extract from control rats. Lane 3: Retinal extract fromexperimental rats. Lane 4: Extract from the optic nerve of control rats.Lane 5: Extract from the optic nerve of experimental rats. Lane 6:Recombinant �-synuclein was expressed using pTrcHis expressionvector (Invitrogen, La Jolla, CA) and purified according to the manu-facturer’s recommendations. Purified �-synuclein contains six histidineresidues attached to the N-terminus of the protein. Lane 7: Extractfrom control A7 rat astrocytes. Lane 8: Extract from A7 rat astrocytessubjected to elevated continuous hydrostatic pressure as described inMaterials and Methods. B: Extracts from rat brain (lane 1), retina (lanes2, 3), or optic nerve (lanes 4, 5) were prepared as described previously(Surguchov et al., 2001a) and subjected to electrophoresis in 12%PAGE, Western blotted, and probed with antibody to �-tubulin. Lane2: Retinal extract from control rats. Lane 3: Retinal extract fromexperimental rats. Lane 4: Extract from the optic nerve of control rats.Lane 5: Extract from the optic nerve of experimental rats. Lane 6:Extract from control A7 rat astrocytes. Lane 7: Extract from A7 ratastrocytes subjected to elevated continuous hydrostatic pressure as de-scribed in Materials and Methods. C: Extracts from rat brain (lane 1),retina (lanes 2, 3), or optic nerve (lanes 4, 5) were prepared as describedpreviously (Surguchov et al., 2001a) and subjected to electrophoresis in12% PAGE, Western blotted, and probed with antibody to actin. Lane2: Retinal extract from control rats. Lane 3: Retinal extract fromexperimental rats. Lane 4: Extract from the optic nerve of control rats.Lane 5: Extract from the optic nerve of experimental rats. Lane 6:Recombinant �-synuclein. Lane 7: Extract from control A7 rat astro-cytes. Lane 8: Extract from A7 rat astrocytes subjected to elevatedcontinuous hydrostatic pressure as described in Materials and Methods.
Fig. 6. �-Synuclein localization in a rat A7 astrocyte culture. A: Con-trol astrocyte culture. B: Astrocyte culture incubated at elevated hy-drostatic pressure. Nuclear localization and association with centro-somes (arrows) are shown. Red, microtubules; green, �-synuclein.
104 Surgucheva et al.
staining (Buchman et al., 1998b). On the other hand,distortion of the neurofilament network is common inneurodegenerative diseases in general and in glaucoma inparticular (Vickers et al., 1995b). 2) Another hypotheticalmechanism of �-synuclein involvement in glaucomatousalterations in the optic nerve is related to its role in thesignal transduction pathway. �-Synuclein activates Elk-1and MAPK (Surguchov et al., 1999, 2001b), and thus itsaccumulation in glial inclusions described here may causeinterruption of such signaling. 3) According to our pre-liminary results, �-synuclein activates several types of ma-trix metalloproteinases (MMPs), enzymes that play animportant role in remodeling of the extracellular matrixand possibly are involved in glaucomatous changes in theoptic nerve (Agapova et al., 2001).
The data on the role of synucleins in neurodegen-eration and their effect on cell survival are controversial.They concern almost exclusively �-synuclein, and in themajority of studies a toxic effect of this member of thesynuclein family has been seen (El-Agnaf et al., 1998;El-Agnaf and Irvine, 2000; Iwata et al., 2001). The level oftoxicity depends on the aggregation of �-synuclein andformation of fibrils (El-Agnaf and Irvine, 2000) and isincreased in the presence of two point mutations describedfor this protein (Kanda et al., 2000). In other studies,�-synuclein supposedly is not involved in neurodegenera-tion and apoptotic death but rather is implicated in thecompensatory response with concomitant survival(Kholodilov et al., 1999), chaperon activity (Ostrerova etal., 1999; Souza et al., 2000a), or even a protective effect(O’Malley and Jensen, 2000). The level of synuclein tox-icity and its effect on cell viability most probably dependnot on a single factor but on a combination of severalfactors, modifying the structure, i.e., 1) posttranslationalmodifications, including nytrosylation (Souza et al.,2000b); 2) the level of dimerization-oligomerization (El-Agnaf et al., 1998); and 3) the presence of mutations(El-Agnaf et al., 1998). For some of the heat shock pro-teins it has been shown that their dimerization stronglydiminishes their chaperone activity (van de Klundert et al.,1998).
In the majority of previous publications concerningthe role of synucleins in neurodegenerative diseases, animportant role of �-synuclein has been documented.Here, we suggest a possible �-synuclein involvement inglaucomatous alterations in the optic nerve, and in aprevious publication we demonstrated a redistribution of�-synuclein immunoreactivity in the retina of AD patients(Surguchov et al., 2001a).
The results presented here do not point to elevatedpressure as a major factor mediating the role of �-synucleinin the changes observed in the glaucomatous optic nerve.Thus, pressure-independent factors not yet identified mayaccount for these changes. Indeed, similar alterations in thepattern of �-synuclein staining were observed in patientswith elevated and normal IOP (compare, for example,panel D with panels C, E, F in Figs. 1 and 2). In addition,we did not find up-regulation of �-synuclein as a result of
elevated pressure (as one would anticipate for a stressprotein or a protein possessing chaperone activity). On thecontrary, a reduction of its immunoreactivity was foundboth in the optic nerve and in astrocytes incubated underelevated pressure. These data may reflect the fact thatelevated pressure is only one of several important factors inthe etiology of glaucoma. However, the similarity ofchanges in immunoreactivity observed in different groupsof glaucoma patients suggests that �-synuclein may poten-tially be considered as a marker of glaucomatous changesin the optic nerve. Overall, these findings point to theimportance of further studies of the �-synuclein role inocular diseases and other pathologies.
ACKNOWLEDGMENTSWe thank Dr. M.R. Hernandez for providing us
with an experimental pressure chamber and for humanocular tissue sections. We express our gratitude to Dr. H.Geller for the A7 astrocyte culture.
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